caa a22 4500 605506841 CHVBK 20210128100629.0 cr unu---uuuuu 210128e20150101xx s 000 0 eng 10.1007/s00775-014-1209-3 doi (NATIONALLICENCE)springer-10.1007/s00775-014-1209-3 The unique serine/threonine phosphatase from the minimal bacterium Mycoplasma synoviae [Elektronische Daten] biochemical characterization and metal dependence [Angela Menegatti, Javier Vernal, Hernán Terenzi] Serine/threonine protein phosphatases have been described in many pathogenic bacteria as essential enzymes involved in phosphorylation-dependent signal transduction pathways and frequently associated with the virulence of these organisms. An inspection of Mycoplasma synoviae genome revealed the presence of a gene (prpC) encoding a putative protein phosphatase of the protein phosphatase 2C (PP2C) subfamily. Here, we report a complete biochemical characterization of M. synoviae phosphatase (PrpC) and the particular role of metal ions in the structure-function relationship of this enzyme. PrpC amino acid sequence analysis revealed that all the residues involved in the dinuclear metal center and the putative third metal ion-coordinating residues, conserved in PP2C phosphatases, are present in PrpC. PrpC is a monomeric protein able to dephosphorylate phospho-substrates with Mn2+ ions' dependence. Thermal stability analysis demonstrated the enzyme stability at mild temperatures and the influence of Mn2+ ions in this property. Mass spectrometry analysis suggested that three metal ions bind to PrpC, two of which with an apparent high-affinity constant. Mutational analysis of the putative third metal-coordinating residues, Asp122 and Arg164, revealed that these variants exhibited a weaker binding of manganese ions, and that both mutations affected PrpC phosphatase activity. According to these results, PrpC is a metal-dependent protein phosphatase member with an improved stability in the holo form and with Asp122, possibly implicated in the third metal-binding site, essential to catalytic activity. SBIC, 2014 PP2C-like phosphatase nationallicence Metal binding nationallicence Mn2+ ion nationallicence Mycoplasma synoviae nationallicence PrpC nationallicence ESI-MS : Electrospray ionization-mass spectrometry nationallicence HAD : Haloacid dehalogenase nationallicence MALDI-TOF/TOF : Matrix-assisted laser desorption/ionization-time-of-flight nationallicence Mw : Molecular weight nationallicence FCP/SCP : [TFIIF(transcription initiation factor IIF)-associating component of CTD (C-terminal domain) phosphatase/small CTD phosphatase] nationallicence WT : Wild type nationallicence Menegatti Angela Departamento de Bioquímica-CCB, Centro de Biologia Molecular Estrutural, Universidade Federal de Santa Catarina, 88040-900, Florianópolis, SC, Brazil aut Vernal Javier Departamento de Bioquímica-CCB, Centro de Biologia Molecular Estrutural, Universidade Federal de Santa Catarina, 88040-900, Florianópolis, SC, Brazil aut Terenzi Hernán Departamento de Bioquímica-CCB, Centro de Biologia Molecular Estrutural, Universidade Federal de Santa Catarina, 88040-900, Florianópolis, SC, Brazil aut JBIC Journal of Biological Inorganic Chemistry Springer Berlin Heidelberg 20/1(2015-01-01), 61-75 0949-8257 20:1<61 2015 20 775 https://doi.org/10.1007/s00775-014-1209-3 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00775-014-1209-3 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Menegatti Angela Departamento de Bioquímica-CCB, Centro de Biologia Molecular Estrutural, Universidade Federal de Santa Catarina, 88040-900, Florianópolis, SC, Brazil aut NATIONALLICENCE 700 1- Vernal Javier Departamento de Bioquímica-CCB, Centro de Biologia Molecular Estrutural, Universidade Federal de Santa Catarina, 88040-900, Florianópolis, SC, Brazil aut NATIONALLICENCE 700 1- Terenzi Hernán Departamento de Bioquímica-CCB, Centro de Biologia Molecular Estrutural, Universidade Federal de Santa Catarina, 88040-900, Florianópolis, SC, Brazil aut NATIONALLICENCE 773 0- JBIC Journal of Biological Inorganic Chemistry Springer Berlin Heidelberg 20/1(2015-01-01), 61-75 0949-8257 20:1<61 2015 20 775